cancers

Review

A Comprehensive Review of Cancer MicroRNATherapeutic Delivery Strategies

Alexis Forterre 1, Hiroaki Komuro 2 , Shakhlo Aminova 3,4 and Masako Harada 4,5,*1 UMR DIATHEC, EA 7294, Centre Européen d’Etude du Diabète, 67200 Strasbourg, France;

a.forterre@ceed-diabete.org2 Department of Cardiovascular Physiology, Tokyo Medical and Dental University, Tokyo 113-8510, Japan;

hkomcvp@tmd.ac.jp3 Lyman Briggs College, Michigan State University, East Lansing, MI 48825, USA; aminovas@msu.edu4 Institute for Quantitative Health Sciences and Engineering (IQ), Michigan State University,

East Lansing, MI 48824, USA5 Department of Biomedical Engineering, Michigan State University, East Lansing, MI 48824, USA* Correspondence: mashar@msu.edu; Tel.: +1-517-884-6940

Received: 22 June 2020; Accepted: 7 July 2020; Published: 9 July 2020�����������������

Abstract: In the field of molecular oncology, microRNAs (miRNAs) and their role in regulatingphysiological processes and cancer pathogenesis have been a revolutionary discovery over the lastdecade. It is now considered that miRNA dysregulation influences critical molecular pathwaysinvolved in tumor progression, invasion, angiogenesis and metastasis in a wide range of cancertypes. Hence, altering miRNA levels in cancer cells has promising potential as a therapeuticintervention, which is discussed in many other articles in this Special Issue. Some of the mostsignificant hurdles in therapeutic miRNA usage are the stability and the delivery system. In thisreview, we cover a comprehensive update on the challenges and strategies for the development oftherapeutic miRNA delivery systems that includes virus-based delivery, non-viral delivery (artificiallipid-based vesicles, polymer-based or chemical structures), and recently emerged extracellular vesicle(EV)-based delivery systems.

Keywords: microRNA delivery; cancer therapy; therapeutic RNA; extracellular vesicles

1. MicroRNA Overview

The central dogma of molecular biology was no longer “central” after the discovery that geneexpression involves more complex layers of regulation than the DNA sequences. MicroRNAs (miRNAs)are one of the most well-studied non-coding RNAs that play a critical role in gene regulation, some ofwhich are dysregulated in association with certain cancer types. The first cancer-associated miRNAwas discovered from the study on the commonly deleted region 13q14 in B-cell chronic lymphocyticleukemia (B-CLL), where the minimally deleted region among patients located at first exons of twonon-protein coding RNA transcripts, DLEU 1 and DLEU 2 (Deleted In Lymphocytic Leukemia 1 and 2),aligned in opposite directions [1]. In 2002, Calin and his colleagues identified two miRNAs, miR-15aand miR-16-1, located within the intron of the DLEU2 gene that was deleted or downregulated in themajority of CLL patients [2]. Further studies revealed that these miRNAs function as tumor suppressorsto induce apoptosis by repressing the B-cell lymphoma 2 (Bcl-2) gene, and inhibit the cell cycle by therepression of cyclin-related genes such as CCND1 and CCNE1 [3,4]. These discoveries sparked thefield of miRNA-cancer research, the exploration of a unique miRNA transcriptome for each cancertype, empowering the better classification of specific cancer types and prognostic information.

The canonical pathway of miRNA biogenesis initiates from a capped and polyadenylated longprimary transcript (pri-miRNA or pri-miR) transcribed by RNA polymerase II (Pol II), resembling that of

Cancers 2020, 12, 1852; doi:10.3390/cancers12071852 www.mdpi.com/journal/cancers

Cancers 2020, 12, 1852 2 of 21

protein-coding transcripts [5]. A stem-loop structure of pri-miRNA is recognized and further processedby the nuclear RNaseIII enzyme—a double-stranded RNA binding domain protein (DROSHA-DGCR8)complex—into a hairpin-loop structure, called precursor-miRNA (pre-miRNA or pre-miR) [5,6].Pre-miR is exported from the nucleus by the Exportin-5-Ran-GTP axis and further processed into themature form of double-stranded RNA by Dicer-TRBP complex [7]. RNA-induced silencing complex(RISC) containing Argonaute (Ago) proteins incorporates one strand of the miRNA duplex and guides itto the partially complementary site within 3’UTR of the target gene [8]. There are several non-canonicalpathways of miRNA biogenesis including a miRNA derived from an intronic region of mRNA bysplicing (Miltron) independent of Drosha processing, and a miRNA trimmed by poly(A)-specificribonuclease (PARN) independent of Dicer cleavage [9,10]. Nevertheless, the vast majority of miRNAsfall into the former category of biogenesis, in which processes and machinery are highly conservedacross evolutionarily distant species.

There were reports on correlations between the dysregulation of miRNA machinery and diseaseprogression and poor prognosis in several tumor types such as ovarian cancer [11], neuroblastoma [12]and lung cancer [13]. In most cases, cancer cells exhibit a distinct miRNA pattern that contributes tospecific traits of malignancies, possibly because other RNA species share the same regulatory pathway.MiRNA binding through a partial complementary sequence (seed sequence) within the target mRNA3’UTR primarily inhibits translation or promotes its degradation. A specific miRNA can regulatehundreds of genes, but in reality, only 30–40% of genes are regulated by miRNAs and the target sites areoften selectively conserved within the 3’UTR [14,15]. There are a couple of reports on miRNA-mediatedgene activation or miRNA binding to non-3’UTR region, which appear as infrequent cases [16].

MiRNA regulation: Genomic loci of miRNA can be either intergenic when they have theirown promoter driving their expression or intragenic where the miRNA gene is within the intron orexon of a protein-coding gene. According to the integrating miRNA inter- and intragenic database(miRIAD), of 1871 human miRNAs, 1072 (57.3%) are intragenic and 799 (42.7%) are intergenic [17].The transcriptional regulation of miRNA genes is much like that of protein-coding genes, determinedby factors including transcription factors, DNA methylation, mutations, copy number variations,RNA stability and cleavages [18–20]. One such example is when glucocorticoid, one of the commonlyused steroid hormones to treat hematopoietic malignancies, binds to its receptor, and the receptorcomplex binds to the upstream promoter region of the miR-17-92 cluster gene resulting in transcriptionalrepression [21]. On the contrary, miRNAs’ specific regulatory mechanism involves miRNA biogenesisor processing machineries, often leading to a global miRNA alteration associated with human cancers.Such dysregulation of RNaseIII type enzymes, nuclear DROSHA and cytoplasmic DICER, as wellas components of the RISC complex, TARBP2 and AGO2, were reported in association with varioustypes of cancers [22]. The emerging role of the growth-promoting tumor microenvironment involvesintra-cellular miRNA regulation [23]. Recent research reported that miRNAs play key roles in thecrosstalk between cancer-associated fibroblasts (CAFs) and tumor cells, which were transferredby cell-derived extracellular vesicles. The extracellular miRNAs from cancer-associated fibroblastsgranted favorable traits such as growth promotion, survival and drug resistance in various tumortypes, including colorectal cancer, breast cancer and pancreatic cancer [24–27]. In summary, the finetuning of miRNA levels is comprehensively coordinated by both internal and external regulatorymechanisms [28].

2. Altered MicroRNA Expression in Cancer Cells

2.1. Altered miRNA Profile in Malignancies

Over the past decade, it has come to light that miRNAs present altered expression patterns inmalignant tissues and cells. Some miRNAs regulate essential genes for cellular homeostasis in whichthe alteration will result in abnormal biological changes, including uncontrolled cell proliferation,angiogenesis, metabolism and apoptosis, leading to malignant formation. All of the key cancer pathways

Cancers 2020, 12, 1852 3 of 21

have an association with miRNA alterations; sensitivity to growth signals (e.g., let-7 family, miR-21);insensitivity to antigrowth signals (e.g., miR-17-92 cluster, miR-195); apoptosis escape (e.g., miR-34a,miR-185, miR-15/miR-16); angiogenesis (e.g., miR-210, miR-26, miR-15b, miR-155); invasion andmetastases (e.g., miR-10b, miR-31, miR-200 family, miR-21, miR-15b); evading immune destruction(e.g., miR-124, miR-155, miR-17-92); tumor-promoting inflammation (e.g., miR-23b, miR-155, let-7d);and genomic instability (e.g., miR-21, miR-155, miR-15b) [29–31]. The malignant transformation willalter cell- and cellular state-specific miRNA expression profiles in healthy tissues. Furthermore, miRNAsare capable of driving malignant formation either by repressing tumor suppressor genes or increasingoncogene expression, thus emphasizing the importance of miRNAs in malignant transformation andas biomarkers to further subclassify cancer types [32–34].

Comprehensive miRNA expression analyses revealed a set of miRNAs that show distinctexpression patterns in various cancer types, which led to biomarker discovery, or further functionalvalidation of these miRNAs. For example, global miRNA expression analysis of diffuse large B-celllymphoma (DLBCL) revealed a reduced level of miR-150 and miR-29b [35,36]. Following this finding,Xiao et al. identified that miR-150 targets transcription factor c-Myb, essential in the development oflymphocytes [37], and Li et al. showed that miR-29b regulates protein kinase B (Akt) which promotessurvival and growth through the PI3K/Akt signaling pathway [38]. Therefore, comprehensive miRNAexpression analysis of cancer cells holds great potential for therapeutic target and biomarker discovery.

Tumor-related miRNAs are broadly classified into two groups: Oncogenic miRNAs (OncomiRs),and Tumor Suppressor miRNAs (TS-miRNAs, TS-miRs). Most, if not all, types of cancers display aninduced expression of some oncomiRs, which promote tumorigenesis by blocking the translation oftumor-suppressive mRNAs (e.g., miR-17-22, miR-125b, and miR-125) [39–41], whereas the reducedexpression of TS-miRNAs (e.g., miR-133a, miR-145, and miR-143) directly inhibits the translation ofoncoproteins [42,43]. Thus, miRNA expression versatility emphasizes their utility in cancer therapeuticstrategies. Therefore, antisense-miRNA (anti-miRs) or miRNA mimics were developed for miRNAtherapy and are widely used to repress oncomiRs or to restore TS-miRs, respectively [44–46].

MiRNAs are found in both intracellular and extracellular regions of the body. Recently, several studieshave revealed the potential of circulating miRNAs as a new class of biomarkers. For example, both the tissueand sera of oral cancer patients presented induced elevated levels of miR-31, which target NAD-dependentdeacetylase SIRT3 that regulates metabolism and energy production [47,48]. Both tissue and sera from gastricand germ cell cancer patients exhibit elevated miR-371-3, which promotes tumor growth and metastasis bydirectly targeting tumor-suppressor gene TOB1 (transducer of ERBB2, 1) [49,50]. Additionally, chemotherapyresistance-related miRNA alteration was reported, miR-155 and miR-221/miR-222 overexpression wasassociated with a poor outcome in lung cancer and poor therapeutic outcomes of anti-estrogenic therapies(Tamoxifen, Fulvestran), respectively [51]. The implication of miRNAs in diagnosis and therapy differsover a wide range of cancer types.

2.2. MiRNA Editing as Cancer Therapy

Certain miRNAs are master regulators of tumorigenesis in some cancers, and thus representpowerful therapeutic targets.

For example, supplementing the reduced miR-29b, the regulator of cell growth, proliferation,and angiogenesis, caused tumor reduction in xenograft mice of breast and gastric cancers [38,52].MiR-34a is another therapeutic miRNA that is generally under-expressed in many cancer types, notablybreast cancer. Some potent targets of miR-34a include transcription factor, Fos-related antigen (Fra-1) thatregulates apoptosis/proliferation/differentiation, and NAD+ dependent histone deacetylase Sirtuin-1(SIRT1) that deactivates tumor suppressor p53 [53,54]. A phase I clinical trial using miR-34a showedantitumor activity in a subset of patients with refractory advanced solid tumors with pre-dexamethasonetreatment, which is discussed in more detail later in this review [55].

The dysregulation of the miR-15a and miR-16-1 cluster involves multiple oncogenic phenotypessuch as cell survival, proliferation, and invasion by directly targeting CCND1 (cyclin D1), WNT3A,

Cancers 2020, 12, 1852 4 of 21

and BCL2, evident in prostate and pancreatic cancers. WNT3A is part of the Wnt/beta-catenin signalingpathway responsible for cell–cell adhesion, and the promotion of oncogenes like c-Myc and CCND1.Thus, miR-15a re-expression showed a decreased viability of pancreatic tumor cells, and its knockdownresulted in an increased invasion and proliferation in prostate cancer in vivo models [56,57].

While the role of miRNA as a regulator of various physiological processes is evident, challengeslie within its delivery. In a body, crude miRNAs are unstable due to various ribonucleases in the blood,if not cleared by phagocytosis through the reticuloendothelial system (RES). In addition, crude miRNAsare unable to cross the cell membrane or the vascular endothelium due to their negative charges.To overcome these problems, various approaches were examined to deliver miRNAs. We will provideexamples of in vivo experimental approaches to modulate miRNA levels across multiple tumor modelsfor therapeutic applications in the following sections.

2.3. MiRNA Inhibition Therapies for OncomiRs

Reducing oncomiRs, frequently overexpressed in human cancers, allows for the restorationof target tumor-suppressor expression that could work as a therapeutic strategy. Commonly usedmiRNA inhibitors, single-stranded antisense, anti-miR oligonucleotides (AMOs), locked nucleicacid (LNA) anti-miRs, antagomiRs, miRNA sponges and small molecule inhibitors of miRNAs(SMIRs), interfere with miRNA biogenesis or block miRNA-mRNA interaction [31,58–61]. For instance,the intravenous injection of antagomiRs against miR-16, miR-122, miR-192, and miR-194 significantlyreduced their endogenous expression without provoking an immune response [62]. Another exampleis with the oncogenic miR-21 antagonist, repressing AKT and subsequent Mitogen-activated proteinkinase (MAPK) pathway activation, which hampered Epithelial–mesenchymal transition (EMT) andangiogenesis in breast cancer [63–65]. MiRNA sponges (RNA molecules with repeated miRNA bindingsequences that can sequester the miRNAs of choice) successfully repress miR-23b expression bothin vitro and in vivo, reducing glioma angiogenesis, invasion, and migration [66].

2.4. MiRNA Replacement Therapies for Tumor-Suppressor MiRNAs

Supplementing tumor-suppressive miRNAs in cancer cells is another therapeutic approach formiRNA alteration. The use of synthetic double-stranded miRNA mimics, pre-miR, or plasmid-encodedmiRNA genes compensates for lost tumor-suppressor miRNAs in order to target tumor-promotingmRNAs [31,44]. The miR-34 family, defective in about half of human cancers, plays a critical role inthe suppression of tumor development [67]. The delivery of miR-34 mimics to cancer cells showed agrowth inhibitory effect, and confirmed the promising use of miRNA mimics in cancer treatment [67].However, the challenges for miRNA therapy need to be addressed. As previously mentioned, miRNAsare unstable in the body due to multiple ribonucleases and RES clearance. Moreover, their negativecharges impede the crossing of the cell membrane or the vascular endothelium. Even though they reachinside of a cell, they are subject to endolysosomal degradation. To ensure effective treatment in cancercell-specific delivery, it is crucial not to disrupt the healthy tissue [68]. Impaired blood perfusion intumors lowers systemic delivery of miRNAs, while the tumor microenvironment acts as a barrier anddisrupts efficient miRNA delivery [69,70]. Tumor-associated immune cells (macrophages, neutrophilsand monocytes) in the tumor microenvironment can nonspecifically uptake miRNAs encapsulated inthe delivery system [65,71]. To overcome these problems, researchers have tried different approachesin delivering therapeutic miRNA mimics or inhibitors.

In the following sections, we will provide several examples to modulate miRNA levels, using in vivomodels for therapeutic applications. Table 1 shows the summary of miRNA mimics or inhibitors testedin vivo via different delivery methods.

Cancers 2020, 12, 1852 5 of 21

Table 1. The Summary of MicroRNA (miRNA) Mimics or Inhibitors Tested in Vivo.

Delivery Systems miRNAs miRNA Type Drug Delivery Route Target Disease Target Gene Ref

Lipid-based systemCationic liposome miR-29b mature-miR - Tail-vein Lung cancer CDK6, DNMT3B, MCL1 [72]Cationic liposome miR-7 pre-miR - Intratumoral Lung cancer IRS-1, RAF-1, EGFR [73]Neutral liposome miR-34a pre-miR - Intravenous Lung cancer BCL-2, c-Met [74]

Neutral liposome let-7miR-34a mimic-miR - Intravenous Lung cancer KRAS [75]

Ionizable liposome miR-200c plasmid - Subcutaneous Lung cancer PRDX2, GAPB/Nrf2, SESN1 [76]

Polymer-based systemPEI-PEG miR-34a dsRNA - Tail-vein Hepatocellular carcinoma SNAI1 [77]

Polyurethane-PEI miR-145 plasmid - Intracranial Glioblastoma Oct4, Sox2 [78]

PEI miR-145miR-33a dsRNA - Intraperitoneal

Intravenous Colon carcinoma c-Myc, ERK5 [79]

Polymeric micelle miR-205 mimic-miR Gemcitabine Intratumoral Pancreatic cancer ZEB-1, SIP-1, HRAS, LRP-1, CAV-1, E-CAD [80]Polymer micelle anti-miR-21 dsRNA Doxorubicin Intratumoral Glioma PTEN [81]PACE polymer anti-miR-21 dsRNA Temozolomide Intracranial Glioblastoma PTEN [82]

PEI miR-203 dsRNA - Subcutaneous Basal cell carcinoma c-JUN [83]

Inorganic-based systemCarbonate apatite miR-4711-5p mimic-miR - Intravenous Colon cancer KLF5, TFDP1 [84]Carbonate apatite miR-4689 mature-miR - Intravenous Metastatic colorectal cancer KRAS, AKT1 [85]Carbonate apatite miR-29b mimic-miR - Intravenous KRAS-mutant colorectal cancer BCL-2, MCL1 [86]

Extracellular Vesicles-based systemExosome miR-143 BP-miR Intravenous Colon cancer - [87]Exosome miR-146b plasmid - Intratumoral Glioma - [88]Exosome miR-145 dsRNA - Tail-vein Lung cancer CDH2 [89]

Exosome miR-122 plasmid Sorafenib IntratumoralIntraperitoneal Hepatocellular carcinoma ADAM10, IGF1R, CCNG1 [90]

Exosome-GE11 peptide let-7 mimic-miR - Intravenous Breast Cancer HMGA2 [91]

Other BiomaterialsAtelocollagen miR-34a pre-miR Subcutaneous Colon cancer E2F [92]Atelocollagen miR-16 pre-miR Intravenous Prostate cancer CDK1, CDK2 [93]Atelocollagen miR-15, miR-16-1 miR with 2′-fluoro - Prostate cancer BCL-2, CCND1, WNT3A [94]

Cancers 2020, 12, 1852 6 of 21

3. Approaches for MiRNA Therapeutic Delivery

3.1. Local Delivery

Due to its bioavailability and reduced toxicity, the intratumoral injection of miRNA mimics or inhibitorsare significantly more effective than those delivered by systemic routes. However, local administration islimited to the localized and readily accessible primary solid tumors such as melanoma, breast and cervicalcancers. One advantage of local delivery is minimal nonspecific uptake by healthy organs, which can leadto unwanted toxicity and immunogenicity.

Halle et al. presented the anti-tumor effect of intratumoral convection-enhanced delivery (CED) ofa 2′-O-methoxyethyl anti-miR-let-7a in combination with phosphodiester/phosphorothioate backboneusing a highly invasive glioblastoma (GBM) orthotopic xenograft model. Importantly, no animalsshowed signs of severe adverse effects while retaining the functional anti-miR effect following onemonth of anti-miR administration [95].

Trang et al. showed that both the intratumoral and intravenous administration of let-7a mimics ledto decreased tumor size in the non-small-cell lung cancer (NSCLC) mouse model [75,96]. In hepatomaxenograft models, intratumoral administration of cholesterol-conjugated 2′-O-methyl-modified miR-375mimics suppressed tumor growth by targeting the AEG-1 gene (astrocyte elevated gene-1) [97].The same group performed intranasal delivery of the lentiviral vector expressing let-7a using theaggressive human NSCLC xenograft model, resulting in an increased let-7 expression followed bythe growth inhibition of KRAS-dependent lung tumors. Additionally, the local delivery of syntheticlet-7b using a polymer-based delivery triggered a specific inhibitory response leading to tumorreduction by 60–70% [96]. The polyethylenimine (PEI)-mediated local application of miR-145 achieveda significant anti-tumor effect in colon carcinoma mouse models [79]. Lastly, Nanoparticles -mediatedintratumoral delivery of siRNA specific to DCAMKL-1 induced expression of let-7a and miR-144 andsubsequent repression of proto-oncogenes c-Myc and Notch-1 in colorectal cancer xenografts, thusresulting in tumor growth inhibition [98]. Local administration is limited to accessible tumors; thus,the development of a systemic delivery approach is necessary to treat other types of cancers andmetastatic tumors.

3.2. Systemic Delivery

The ideal delivery system should protect miRNAs from early degradation in the bloodstream,carry them to the target cells, and facilitate cellular uptake without inducing any immunogenicresponse [99,100]. To this end, oligonucleotide editing and systemic miRNA delivery systemswere explored as improved therapeutic deliveries of miRNA mimics and inhibitors. The chemicalmodification of miRNA oligonucleotides enhances miRNA stability and prevents their degradation bynucleases in the circulation. For example, an addition of 2′-O-methyl (2′-O Me), 2′-O-methoxyethyl or2′-fluoro to the 2′-OH in riboses, enhances the stability and binding affinity of anti-miR, and provideslong-lasting effects in the liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries andadrenals [101]. LNAs are another type of chemically modified RNA nucleotide analog with highnuclease resistance, applicable to siRNA and miRNA mimics/inhibitors. The systemic administrationof the LNA-anti-mir-122 showed a dose-dependent downregulation of liver-specific miR-122 levels inmice liver for several weeks, and the upregulation of a set of target mRNAs [102]. LNA-anti-miR-122prevents hepatitis C virus (HCV) replication, and is the first miRNA drug to successfully enter Phase IItrials for the treatment of HCV infection. In turn, this represents a promising preventive therapy forhepatocellular carcinoma (HCC) resulting from HCV infection [103]. However, such modified miRNAshave short half-lives because of the rapid renal and hepatic clearances resulting in limited tumor uptakeand biodistribution [65]. In 2016, Teplyuk et al. tested three delivery routes for the miR-10b antisenseoligonucleotide inhibitors (ASO): 1) direct intratumoral injections, 2) continuous osmotic delivery and3) systemic intravenous injections in intracranial human glioma-initiating stem-like cells (GSC)-derivedxenograft and murine GL261 allograft models in athymic and immunocompetent mice, both resulting

Cancers 2020, 12, 1852 7 of 21

in the miR-10b target genes (i.e., MBNL1-3, SART3, and RSRC1) increased expression thus inducingthe reduction of growth and progression of intracranial GBM with no significant systemic toxicitywhen using the ASO local or systemic administrations [104].

Viral (3.3) and non-viral (3.4) vectors are commonly used vectors for miRNA delivery [41,105].Although viral vectors are popular due to their efficiency, the adverse effects from immune responses areinevitable. Thus, non-viral vectors are the preferred choice for clinical studies. Moreover, the formulationof miRNAs in nanoparticles is an alternative strategy investigated by many groups [106].

3.3. Viral Delivery

Viral vectors transfer pre-miR or mature miRNA cloned in a plasmid to the tumor cells,generate mature miRNA, drive expression with the viral promoter, and subsequently repress and/ordegrade target mRNAs [41]. Lentivirus, adenovirus and adeno-associated virus (AAV)-mediateddelivery systems have successfully delivered either miRNA mimics or antagonists to the nuclei of tumorcells, followed by miRNA expression and function [107]. Kasar et al. showed the lentiviral delivery ofmiR-15a/16 to a New Zealand Black (NZB) mouse model of chronic lymphocytic leukemia (CLL) restoredexpression of these miRNAs whose expression were lacking in CLL [108]. Another study showed theintravenous administration of lentivirus containing miR-494 antagonists, which reduced the activity ofmyeloid-derived suppressor cells (MDSCs) through miR-494 inhibition, which is pro-angiogenesis andpromotes tumor growth, in a murine breast cancer model [109]. Genetic manipulation allows for theconjugation of targeting moieties to the viral capsid proteins, which improves the affinity between viralvectors and cancer-specific receptors for specific delivery into the tumors [65]. Nevertheless, one shouldkeep in mind that lentiviruses integrate their viral DNA into the host genome, which could lead tomutagenesis and the activation of oncogenic pathways. In contrast, AAVs are an attractive alternativefor therapeutic delivery because of their nature, keeping their genomes in an episomal form. Kota et al.showed that the AAV-mediated delivery of miR-26a, known for its tumor-suppressing property, inan HCC mouse model resulted in the significant reduction of tumor growth without toxicity [68].Although viral vectors can efficiently deliver functional miRNA antagonists or miRNA mimicsinto tumor tissues, the immunogenic response remains a serious concern in clinical applications.Thus, various non-viral delivery methods were developed as a safer alternative for miRNA delivery [110].

3.4. Non-Viral Delivery

Compared to viral delivery, non-viral methods are generally inefficient in miRNA transferwith an even shorter efficacy in target gene repression. However, recent advancements in thefield have improved miRNA delivery by chemical modifications, making them suitable for clinicalapplications [65]. Non-viral vectors are classified broadly into these groups: polymeric vectors (PEIs,polylactic-co-glycolic acid/PLGA, chitosans and dendrimers) [111], lipid-based carriers (neutral/cationicand targeting-modified), biomaterials [112], and inorganic materials (gold, diamond, silica, and ferricoxide) [113].

3.5. Nanoparticles

Among non-viral vectors, nanoparticles are the most common choice in delivering exogenousnucleic acids, including DNA, mRNA, siRNA and miRNA, as seen in recent studies [114,115].Despite their popularity, technical problems, including safety and efficacy, restrict its clinical usageas a therapeutic vector [116]. Nanoparticle-mediated miRNA delivery must overcome hurdles inthe body, such as degradation, rapid renal clearance, endosomal escape, and pharmacokinetics forefficient delivery [110,117,118]. In particular, nanoparticles accumulate in tumors through the enhancedpermeability and retention (EPR) effect, where the enriched targeting highly depends on the size ofnanoparticles [119]. When designing nanoparticles, these problems need to be addressed. The followingsection will introduce some of the improved nanoparticle delivery systems.

Cancers 2020, 12, 1852 8 of 21

3.5.1. Lipid-Based Vectors

Lipid-based vectors are the most widely used nanoparticle for in vivo gene therapy to date,which form spherical structures composed of phospholipid bilayers with an aqueous core encapsulatingnucleic acids-drugs [120]. The advantages of liposome-based nanoparticles include biocompatibility,flexibility, low immunogenicity, and versatility of administration routes. The efficiency depends onphysicochemical properties such as particle size, surface charge and lipid composition. Recent advancesin the understanding and modeling of liposomes have enabled adjustments of their performanceparameters, such as pharmacokinetics and bioavailability, in specific clinical applications that areoptimal for the disease context [121]. For instance, the cationic Lipoplex-based delivery of miR-29mimics and miR-7-encoding plasmid efficiently accumulated in the tumor site and achieved significanttumor reduction in a murine xenograft tumor model of lung cancer (Figure 1a) [72,73]. Neutral liposomeshave improved tumor accumulation as compared to cationic liposomes due to reduced RES clearanceand prolonged circulation [122]. In fact, the neutral liposome-mediated delivery of miR-34a and let-7bmimics facilitated lung tumor-repression without elevating liver and kidney enzymes in serum withoutinducing a nonspecific immune response in the mice [74,75]. Ionizable liposomes (NOV340) are cationicat low pH, and neutral or anionic at neutral or higher pH, and can selectively enhance cellular uptake.This attribute was used to deliver miR-200c resulting in enhanced radiosensitivity of lung cancerin vivo [76]. The ionizable liposome-miR-34 complex (MRX34) entered phase I clinical trials for livercancer and metastasis from other cancers (NCT01829971), which were later halted following multipleimmune-related severe adverse events (SAE) observed in patients dosed with MRX34 over the course ofthe trial [123].

Cancers 2020, 12, x 3 of 24

Lipid-based vectors are the most widely used nanoparticle for in vivo gene therapy to date, which form spherical structures composed of phospholipid bilayers with an aqueous core encapsulating nucleic acids-drugs [120]. The advantages of liposome-based nanoparticles include biocompatibility, flexibility, low immunogenicity, and versatility of administration routes. The efficiency depends on physicochemical properties such as particle size, surface charge and lipid composition. Recent advances in the understanding and modeling of liposomes have enabled adjustments of their performance parameters, such as pharmacokinetics and bioavailability, in specific clinical applications that are optimal for the disease context [121]. For instance, the cationic Lipoplex-based delivery of miR-29 mimics and miR-7-encoding plasmid efficiently accumulated in the tumor site and achieved significant tumor reduction in a murine xenograft tumor model of lung cancer (Figure 1a) [72,73]. Neutral liposomes have improved tumor accumulation as compared to cationic liposomes due to reduced RES clearance and prolonged circulation [122]. In fact, the neutral liposome-mediated delivery of miR-34a and let-7b mimics facilitated lung tumor-repression without elevating liver and kidney enzymes in serum without inducing a nonspecific immune response in the mice [74,75]. Ionizable liposomes (NOV340) are cationic at low pH, and neutral or anionic at neutral or higher pH, and can selectively enhance cellular uptake. This attribute was used to deliver miR-200c resulting in enhanced radiosensitivity of lung cancer in vivo [76]. The ionizable liposome-miR-34 complex (MRX34) entered phase I clinical trials for liver cancer and metastasis from other cancers (NCT01829971), which were later halted following multiple immune-related severe adverse events (SAE) observed in patients dosed with MRX34 over the course of the trial [123].

Figure 1. Potent anti-tumor effects of miRNA therapy through various delivery methods. (a). Local liposome-miR-7-plasmid treatment of xenograft mice [73]. (b) In vivo tumorigenicity assessment of polyurethane-short branch polyethylenimine (PU-PEI)-mediated miR-145 delivery to patient-derived Glioblastoma (GMB) cells [78]. (c) Tumor sizes of xenograft mice after systemic super carbonate apatite (sCA)-mediated miR-4711-5p delivery [84]. (d) Inhibition of metastatic tumor growth in bone tissues by the atelocollagen-mediated miRNA treatment [93]. (e) Inhibition of breast cancer development in vivo using GE11-positive exosomes containing let-7a [91].

3.5.2. Polymer-based Vectors

Polymer-based vectors encompass synthetic biodegradable polymers, copolymer, or natural polymers such as collagen, chitosan and gelatin. Polymer nanoparticle’s advantages include high stability, ease of substitution, the addition of functional groups and flexibility [124]. The characteristics of these polymers such as size, morphology, charge and degradation can be altered by changing the molecular weight, polymer composition, or architecture, including linear, branched, dendrimer and copolymer. PEI, cationic polymer is the most frequently used polymer-based vector for therapeutic gene delivery. Cationic polymers have many amino groups and can use the proton sponge effect to promote escape from endosomal membranes. PEI-mediated miRNA mimics were proven effective in hepatocellular carcinoma by miR-34a, in glioblastoma by miR-145, and in colon cancer by miR-145 and -33 (Figure 1b) [77–79]. Cationic polymer-mediated co-delivery of miRNA

Figure 1. Potent anti-tumor effects of miRNA therapy through various delivery methods. (a) Localliposome-miR-7-plasmid treatment of xenograft mice [73]. (b) In vivo tumorigenicity assessment ofpolyurethane-short branch polyethylenimine (PU-PEI)-mediated miR-145 delivery to patient-derivedGlioblastoma (GMB) cells [78]. (c) Tumor sizes of xenograft mice after systemic super carbonate apatite(sCA)-mediated miR-4711-5p delivery [84]. (d) Inhibition of metastatic tumor growth in bone tissues bythe atelocollagen-mediated miRNA treatment [93]. (e) Inhibition of breast cancer development in vivousing GE11-positive exosomes containing let-7a [91].

3.5.2. Polymer-Based Vectors

Polymer-based vectors encompass synthetic biodegradable polymers, copolymer, or naturalpolymers such as collagen, chitosan and gelatin. Polymer nanoparticle’s advantages include highstability, ease of substitution, the addition of functional groups and flexibility [124]. The characteristicsof these polymers such as size, morphology, charge and degradation can be altered by changing themolecular weight, polymer composition, or architecture, including linear, branched, dendrimer andcopolymer. PEI, cationic polymer is the most frequently used polymer-based vector for therapeutic

Cancers 2020, 12, 1852 9 of 21

gene delivery. Cationic polymers have many amino groups and can use the proton sponge effect topromote escape from endosomal membranes. PEI-mediated miRNA mimics were proven effective inhepatocellular carcinoma by miR-34a, in glioblastoma by miR-145, and in colon cancer by miR-145 and-33 (Figure 1b) [77–79]. Cationic polymer-mediated co-delivery of miRNA mimics and chemotherapeuticdrugs, such as Gemcitabine, Doxorubicin and Temozolomide, have improved therapeutic effects inin vivo tumor models [80–82]. While the results were promising, PEI toxicity, regardless of modifications,derived from excess positive charge, and low biological degradation in serum from non-specific bindingto proteins in vivo, excluded them from a choice of clinical vectors [41].

Polyamidoamine (PAMAM) dendrimers, dendrimer-based polymeric vectors, are hyperbranchedpolymers with high positive surface charge, which increases the toxicity following its administrationdue to hepatic accumulation [125]. Conjugation with negatively charged polyethylene glycol (PEG) canprevent PAMAM toxicity [41]. Chitosan has a profound nucleic acid binding affinity when complexedwith miRNA. MiR-200c in vitro and miR-34a in vivo efficiently delivered into breast cancer cellsand into the bone metastasis model, respectively, resulted in decreased angiogenesis, invasion, EMT,metastasis, and increased apoptosis [126–129]. PLGA (Poly lactic-co-glycolic acid) is a negatively chargedbiodegradable and biocompatible polymeric nanocarrier, approved by the FDA as a drug-deliverysystem and is in phase II clinical trials for siRNA therapeutic delivery (NCT01676259) [110]. Physical andchemical surface manipulations, such as an addition of branched polyesters with modified amine andcationic residues (PVA-PLGA, DEAPA-PLGA, DMAPA) stabilize the nucleic acids in the polymericmatrix in order to further reduce the toxicity and degradation issues [130,131]. Indeed, the formulationof PLGA complexed with positively-charged PEI and anionic hyaluronic acid was found to efficientlydeliver a DNA plasmid-containing miR-145 to colon cancer cells in vivo, thus leading to increasedanti-proliferative and pro-apoptotic activities [132]. Similarly, miR-34a mimics packaged in the PLGAchitosan delivery vector decreased tumor size and improved survival in a multiple myeloma xenograftmodel [133]. Zhou et al. demonstrated that ester-based dendrimer carrier-mediated delivery of let-7gmiRNA mimics significantly suppressed liver tumor growth with low dendrimer-related toxicityin vivo [134]. Shatsberg et al. developed a polymeric nanogel complex with miR-34a mimics for activecytoplasmic delivery, resulting in the inhibition of tumor growth in human U-87 MG GBM-bearingSCID mice, thus emphasizing the therapeutic potential of such strategy for GBM [135].

3.5.3. Inorganic Vectors

Altering the size, shape, and porosity allows for the engineering of inorganic vectors.Among well-studied inorganic vectors, including calcium phosphate, porous silica, gold andcarbon nanotubes, calcium phosphate (CaP) is the most successful vehicle for miRNA gene therapy.CaP, the inorganic component of bone and teeth, is biocompatible, biodegradable, easy to handle anddissolves under acidic pH in endosomes, leading to osmotic swelling and the release of encapsulatedmiRNA within the cell [136]. The CaP-mediated delivery of miR-4711-5p in colon cancer (Figure 1c) [84],miR-4689 in metastatic colorectal cancer [85], and miR-29b in KRAS-mutant colorectal cancer [86] were allshown to be effective. A study using a hydrogel-embedded gold-nanoparticle-based delivery system incombination with cisplatin revealed that the efficient release of miR-96 and miR-182 strongly suppressedmetastasis with significant shrinkage of the primary tumor in a breast cancer mouse model [137].In 2017, T.J. et al. showed a targeted therapy strategy for glioblastoma using folate (FA)-conjugatedto three-way-junction (3WJ)-based RNA nanoparticles (RNP) for the delivery of anti-miR-21 LNA.Following its specific delivery, miR-21 expression was repressed in glioblastoma cells both in vitro andin vivo, thus resulting in antioncogenic PTEN and PDCD4 expression restoration, cell apoptosis andthe regression of tumor growth [138]. Bertucci et al. developed a strategy using mesoporous silicananoparticles (MSNPs) loaded with temozolomide (TMZ), an antineoplastic agent for malignant glialtumor treatment. These nanoparticles conjugated with the polyarginine-peptide nucleic acid R8-PNA,which targets miR-221, induced a pro-apoptotic effect in a temozolomide-resistant T98G cell line [139].

Cancers 2020, 12, 1852 10 of 21

3.5.4. Other Biomaterials

Atelocollagen (ATE), a nanoformulation, prepared by pepsin treatment from type I collagenof calf dermis, is one of the biomaterials with potential application as a gene delivery vector [140].The intratumoral administration of ATE-miR-34a complex into a xenograft model of colon cancerresulted in a significant decrease in tumor size [92]. Similarly, the use of ATE in vivo for the transfectionof synthetic miR-16 into the bone metastasis of prostate cancer efficiently inhibited tumor growth(Figure 1d) [93]. Additionally, the conjugation of miR-15a and miR-16-1 to ATE-RNA aptamer (APT)hybrid to target prostate-specific membrane antigen (PSMA) expressed PCa cells enhanced the strategyefficacy in vitro and in vivo by inducing the killing of cancer cells [94].

3.5.5. Exosome/Extracellular Vesicle-Based Vectors

Lipid-bilayer membrane-enclosed biological nanoparticles, termed exosomes, or extracellularvesicles (EVs), are naturally secreted from cells, and mediate cellular communication within the bodyby transferring nucleic acids, such as DNA, RNA and proteins from one cell to the other [141,142].Because of their potential diagnostic and therapeutic value, the exploitation of EVs for clinicalapplication is under intensive investigation [143–146].

EVs are mainly divided into three categories: exosomes, microvesicles and apoptotic bodies.Exosomes (30–150 nm) originate from the endosomal system, whereas microvesicles (50–1000 nm) shedfrom the plasma membrane. Despite their distinct biogenesis and intracellular origin, there is no apparentdifference between these subpopulations except median size, and there is no precise method to separatethese populations at this stage, thus “EV” is used as a collective term for exosomes and microvesicles ofsizes around 100 nm [141,147–149]. As a natural biomolecular carrier, EV-mediated miRNA delivery hassignificant advantages over other delivery systems. MiRNAs are transported and selectively deliveredto other cell types via surface receptors or proteins present on the surface of recipient cells [150–153].Unlike synthetic nanoparticles, miRNAs delivered by EV are not entrapped by endosomes within thecell (Figure 2). Hence, the miRNAs evade the endosome trap and escape pathway, thus act faster andmore efficiently in target gene repression at the cellular level. EVs can protect encapsulated miRNAfrom RNase degradation or harsh environments, enabling the transportation in bodily fluids [154,155].EV-mediated miRNA delivery was proven effective for delivery of miR-143 in colon cancer [87], miR-146bin glioma [88], and miR-145 in lung cancer [89]. Other groups have demonstrated that exosome-mediatedmiR-122 enhanced the chemosensitivity of Sorafenib in hepatocellular carcinoma [90]. There are severalmethods for conjugation of peptides to EVs membrane that enhances cell specificity [155–157]. Ohno et al.developed EVs conjugated with epidermal growth factor (EGF)–specific peptide (GE11) that deliveredlet-7a to EGFR-expression breast cancer (Figure 1d) [91]. These studies revealed the promising potentialof EVs to bring innovation to current therapeutic delivery approaches. However, methods of massproduction, as well as effective packaging, are yet to be developed.

Cancers 2020, 12, x 5 of 24

intratumoral administration of ATE-miR-34a complex into a xenograft model of colon cancer resulted in a significant decrease in tumor size [92]. Similarly, the use of ATE in vivo for the transfection of synthetic miR-16 into the bone metastasis of prostate cancer efficiently inhibited tumor growth (Figure 1d) [93]. Additionally, the conjugation of miR-15a and miR-16-1 to ATE-RNA aptamer (APT) hybrid to target prostate-specific membrane antigen (PSMA) expressed PCa cells enhanced the strategy efficacy in vitro and in vivo by inducing the killing of cancer cells [94].

3.5.5. Exosome/Extracellular Vesicle-based Vectors

Lipid-bilayer membrane-enclosed biological nanoparticles, termed exosomes, or extracellular vesicles (EVs), are naturally secreted from cells, and mediate cellular communication within the body by transferring nucleic acids, such as DNA, RNA and proteins from one cell to the other [141,142]. Because of their potential diagnostic and therapeutic value, the exploitation of EVs for clinical application is under intensive investigation [143–146].

EVs are mainly divided into three categories: exosomes, microvesicles and apoptotic bodies. Exosomes (30–150 nm) originate from the endosomal system, whereas microvesicles (50–1,000 nm) shed from the plasma membrane. Despite their distinct biogenesis and intracellular origin, there is no apparent difference between these subpopulations except median size, and there is no precise method to separate these populations at this stage, thus “EV” is used as a collective term for exosomes and microvesicles of sizes around 100 nm [141,147–149]. As a natural biomolecular carrier, EV-mediated miRNA delivery has significant advantages over other delivery systems. MiRNAs are transported and selectively delivered to other cell types via surface receptors or proteins present on the surface of recipient cells [150–153]. Unlike synthetic nanoparticles, miRNAs delivered by EV are not entrapped by endosomes within the cell (Figure 2). Hence, the miRNAs evade the endosome trap and escape pathway, thus act faster and more efficiently in target gene repression at the cellular level. EVs can protect encapsulated miRNA from RNase degradation or harsh environments, enabling the transportation in bodily fluids [154,155]. EV-mediated miRNA delivery was proven effective for delivery of miR-143 in colon cancer [87], miR-146b in glioma [88], and miR-145 in lung cancer [89]. Other groups have demonstrated that exosome-mediated miR-122 enhanced the chemosensitivity of Sorafenib in hepatocellular carcinoma [90]. There are several methods for conjugation of peptides to EVs membrane that enhances cell specificity [155–157]. Ohno et al. developed EVs conjugated with epidermal growth factor (EGF)–specific peptide (GE11) that delivered let-7a to EGFR-expression breast cancer (Figure 1d) [91]. These studies revealed the promising potential of EVs to bring innovation to current therapeutic delivery approaches. However, methods of mass production, as well as effective packaging, are yet to be developed.

Figure 2. MiRNA uptake via nanoparticles. The confocal images of Dy547 labeled miRNA mimic (red) delivery to SKBR3 cells by PEI (left), lipofectamine2000 (middle), or EV (right) with DAPI staining (blue) at 24-hr time point. (Images provided by the Harada Lab).

The advantages and disadvantages of each delivery method are shown (Table 2).

Table 2. Advantages and Disadvantages of MicroRNA therapeutic Delivery Systems.

Figure 2. MiRNA uptake via nanoparticles. The confocal images of Dy547 labeled miRNA mimic (red)delivery to SKBR3 cells by PEI (left), lipofectamine2000 (middle), or EV (right) with DAPI staining(blue) at 24-hr time point. (Images provided by the Harada Lab).

Cancers 2020, 12, 1852 11 of 21

The advantages and disadvantages of each delivery method are shown (Table 2).

Table 2. Advantages and Disadvantages of MicroRNA therapeutic Delivery Systems.

Delivery Systems Advantages Disadvantages

Viral Vectors - High gene delivery efficiency - Highly immunogenic

Adenoviral vector- High gene delivery efficiency

- High packaging gene-size capacity- Ability to transfer dividing cell

- Highly immunogenic- Short term transgene expression

Adeno-associated viral vector

- High gene delivery efficiency- Long term transgene expression

- Organ specificity possible (serotype)- Ability to transfer dividing and non-dividing cells

- Low immunogenicity

- Hard production vectors- Low gene-packaging capacity

Lentiviral vector

- High gene delivery efficiency- Long term transgene expression

- Ability to transfer dividing and non-dividing cells- Low immunogenicity

- Potential genomic integration

Lipid-based system

- Ability to functionalize for targeting- Ability to co-deliver gene therapy and chemotherapy

- Controllable size- Systemic gene delivery

- High packaging gene-size capacity- Non-immunogenic

- Transient expression

- Low delivery efficiency in vivo- Nonspecific gene delivery

- Cytotoxicity

Polymer-based system

- Ability to functionalize for targeting- Ability to co-delivery gene therapy and chemotherapy

- Controllable size- Systemic gene delivery

- High packaging gene-size capacity- Non-immunogenic

- Transient expression

- Low delivery efficiency in vivo- Nonspecific gene delivery

- Cytotoxicity

Inorganic-based system

- Ability to functionalize for targeting- Controllable size

- Systemic gene delivery- High packaging gene-size capacity

- Non-immunogenic- Transient expression

- Easy to produce

- Low gene delivery efficiency

Extracellular Vesicle-based system

- Ability to functionalize for targeting- Ability to co-delivery gene therapy and chemotherapy

- Systemic gene delivery- High packaging gene-size capacity

- Non-immunogenic- Highly compatibility

- Low immune clearance- Organ specificity possible

- High stability

- Insufficient studies on EV-based gene therapy- Diverse composition

- Low production

4. MiRNA-Based Therapies in Animal Models and Clinics

4.1. Therapeutic MiRNA Candidates in Preclinical Studies

Several of the miRNA-based therapeutics are currently being tested in preclinical and clinical trials.MiR-10b is a promising target for glioblastoma therapy involved in regulating cell migration, invasion andmetastasis [158]. The clinical significance of miR-10b is its role in metastatic tumors where antagomiR-10breduces metastasis in tumor-bearing mice by restoring Hoxd10 gene expression [158–160]. Over 100 studieson miR-10b with metastatic cancers have revealed its central role in various metastatic tumors [161]. MiR-221is another potent target for metastatic cancer, in which anti-miR-221 caused a significant decrease in thesize and number of tumor nodules in the liver of the transgenic mice model [162]. Cantafio et al. performedpharmacokinetic (PK) and pharmacodynamic (PD) studies, and revealed a short-half-life, optimal tissuebioavailability and minimal urine excretion of LNA-anti-miR-221 (LNA-i-miR-221) along with three-weekspan p27 target upregulation in xenograft tumors. No LNA-i-miR-221 associated toxicity was observed intheir non-human primate study [163]. The loss of miR-16 is associated with a diverse range of tumors,including NSCLC [164], prostate cancer [57], or malignant pleural mesothelioma [165]. Reid et al. usedbacteria-derived minicells to deliver miR-16 mimics in malignant pleural mesothelioma nude mousemodels to show tumor growth inhibition through Bcl-2 and CCND1 targeting [165].

Cancers 2020, 12, 1852 12 of 21

4.2. Clinical Studies Involving MiRNA-Based Therapy

The first siRNA drug approval for clinical use established the groundwork for miRNA drugdevelopment [166]. Mirna Therapeutics, Inc. (Carlsbad, CA, USA) developed anti-miRNA technology,including MRX34, a miR-34 mimic encapsulated in a liposomal nanoparticle formulation (NOV40). It isthe first miRNA mimic to enter clinical development with a focus on patients diagnosed with primaryliver cancer, NSCLC, lymphoma, melanoma, multiple myeloma, or renal cell carcinoma. In parallel,MRX34 administration alone or in combination with radiotherapy (XRT) reduced p53 regulated PDL1expression in non-small-cell lung tumors and antagonized T-cell exhaustion. However, immune-relatedadverse responses led to patient deaths (immune reactions still remains unclear), after which themulticenter phase I trial was halted, and now these preclinical trials are under investigation tounderstand better the immune-related toxicities [55,123,167].

In collaboration with Asbestos Diseases Research Institute (Sydney, Australia), the Australiancompany EnGeneIC started the phase I clinical trial using miR-16 mimic (MesomiR-1) loadedto bacterial-derived nanocells system, which achieved the potent inhibition of tumor growth(NCT02369198). Malignant pleural mesothelioma (MPM) and advanced NSCLC patients, refractoryto standard therapy, were intravenously administered the miR-16 mimic-based EnGeneIC DeliveryVehicle (EDV) complex in which the surface is conjugated with an EGFR-targeting antibody to facilitatetumor site targeting. The first-dose level of 5 billion nanocells loaded with 1.5 µg miR-16 mimics didnot induce an adverse immune response or toxic effects, which allows for the continuation of the phaseII clinical trial [168,169].

Owing to promising preclinical results, MiRagen Therapeutics, Inc. (Boulder, CO, USA) initiatedthe phase I clinical trial of the anticancer LNA anti-miR-155 (MRG-106) efficacy on mycosis fungoides(MF) patients, which is the most common subtype of cutaneous T-cell lymphoma (CTCL) (NCT02580552).In this trial, the reported adverse toxic effects pended the trial to optimize safer therapeutic dosesand specific administration routes [41,145,170]. Table 3 shows a summary of miRNA-based TherapyClinical Trials in the U.S.

Table 3. MiRNA-based Therapy Clinical Trials in the U.S.

Company Name TherapeuticAgent

DeliverySystem Condition or Disease Clinical Phase, Status

miRagenTherapeutics MRG-106 Anti-miR-155 LNA-antisense

Cutaneous T-cell Lymphoma(CTCL)/Mycosis Fungoides

(MF)

Phase II (NCT03837457),Enrolling by invitation

Phase I (NCT03713320),Active, not recruiting

miRagenTherapeutics MRG-106 Anti-miR-155 LNA-antisense

CTCL, MF, ChronicLymphocytic Leukemia (CLL),

Diffuse Large B-cellLymphoma (DVBCL),Activated B-cell (ABC)subtype, Adult T-cell

Leukemia/Lymphoma (ATLL)

Phase I (NCT02580552),Active, not recruiting

MirnaTherapeutics Inc. MRX-34 miR-34 mimic

dsRNAliposomal

nanoparticleMelanoma Phase I (NCT02862145),

Withdrawn

MirnaTherapeutics Inc. MRX-34 miR-34 mimic

dsRNAliposomal

nanoparticle

Primary liver cancer, SCLS,Lymphoma, Melanoma,

Multiple myeloma, Renal cellcarcinoma, NSCLC

Phase I (NCT01829971),Terminated

EnGeneIC MesomiR-1 miR-16 mimic EnGeneICDream Vector

Malignant pleuralmesothelioma,

Non-small-cell lung cancer

Phase I (NCT02369198),Completed

5. Conclusions

The development of strategies aimed to efficiently deliver miRNAs will enhance theeffectiveness of therapeutic interventions. MiRNAs are one of the most promising therapeutic

Cancers 2020, 12, 1852 13 of 21

targets with well-characterized expressions and functions within cancer cells with high specificityto cancer-related pathways. However, their delivery remains a challenge for miRNA therapeutics.The growing effort in the development of delivery vehicles, including nanoparticles, biomaterials and EVs,addresses the obstacles associated with cell-specific targeting, intracellular delivery, miRNA stability andcarrier-associated toxicity. The newly emerged natural delivery system of exosomes/EVs offers enormouspotential as a delivery vehicle in conjunction with the targeting ligand on their surface, which allowsfor a biocompatible delivery system with specificity to cancer cell types. The improvements in miRNAencapsulation, surface engineering and isolation/generation of EVs will advance the development oftargeted-therapeutic miRNA delivery.

Author Contributions: All the authors reviewed the information from the available bibliography, wrote themanuscript and designed the figures. M.H. and A.F. had the idea of writing the review. M.H. coordinated the wholework and revised the final version. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by IQ 2017-2021 “SF” to MH.

Conflicts of Interest: The authors declare no conflicts of interest.

References

1. Liu, Y.; Corcoran, M.; Rasool, O.; Ivanova, G.; Ibbotson, R.; Grander, D.; Iyengar, A.; Baranova, A.; Kashuba, V.;Merup, M.; et al. Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome13q14, frequently deleted in chronic lymphocytic leukemia. Oncogene 1997, 15, 2463–2473. [CrossRef][PubMed]

2. Calin, G.A.; Dumitru, C.D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.; Rattan, S.; Keating, M.;Rai, K.; et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 inchronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 2002, 99, 15524–15529. [CrossRef]

3. Cimmino, A.; Calin, G.A.; Fabbri, M.; Iorio, M.V.; Ferracin, M.; Shimizu, M.; Wojcik, S.E.; Aqeilan, R.I.;Zupo, S.; Dono, M.; et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA2005, 102, 13944–13949. [CrossRef] [PubMed]

4. Lerner, M.; Harada, M.; Loven, J.; Castro, J.; Davis, Z.; Oscier, D.; Henriksson, M.; Sangfelt, O.; Grander, D.;Corcoran, M.M. DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycleinhibitory microRNAs miR-15a and miR-16-1. Exp. Cell Res. 2009, 315, 2941–2952. [CrossRef] [PubMed]

5. Cai, X.; Hagedorn, C.H.; Cullen, B.R. Human microRNAs are processed from capped, polyadenylatedtranscripts that can also function as mRNAs. RNA 2004, 10, 1957–1966. [CrossRef]

6. Lee, Y.; Ahn, C.; Han, J.; Choi, H.; Kim, J.; Yim, J.; Lee, J.; Provost, P.; Radmark, O.; Kim, S.; et al. The nuclearRNase III Drosha initiates microRNA processing. Nature 2003, 425, 415–419. [CrossRef]

7. Hutvagner, G.; McLachlan, J.; Pasquinelli, A.E.; Balint, E.; Tuschl, T.; Zamore, P.D. A cellular function forthe RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 2001, 293,834–838. [CrossRef]

8. Cifuentes, D.; Xue, H.; Taylor, D.W.; Patnode, H.; Mishima, Y.; Cheloufi, S.; Ma, E.; Mane, S.; Hannon, G.J.;Lawson, N.D.; et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalyticactivity. Science 2010, 328, 1694–1698. [CrossRef]

9. Ha, M.; Kim, V.N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014, 15, 509–524. [CrossRef]10. Treiber, T.; Treiber, N.; Meister, G. Regulation of microRNA biogenesis and its crosstalk with other cellular

pathways. Nat. Rev. Mol. Cell Biol. 2019, 20, 5–20. [CrossRef]11. Merritt, W.M.; Lin, Y.G.; Han, L.Y.; Kamat, A.A.; Spannuth, W.A.; Schmandt, R.; Urbauer, D.; Pennacchio, L.A.;

Cheng, J.F.; Nick, A.M.; et al. Dicer, Drosha, and outcomes in patients with ovarian cancer. N. Engl. J. Med.2008, 359, 2641–2650. [CrossRef]

12. Lin, R.J.; Lin, Y.C.; Chen, J.; Kuo, H.H.; Chen, Y.Y.; Diccianni, M.B.; London, W.B.; Chang, C.H.; Yu, A.L.microRNA signature and expression of Dicer and Drosha can predict prognosis and delineate risk groups inneuroblastoma. Cancer Res. 2010, 70, 7841–7850. [CrossRef] [PubMed]

13. Karube, Y.; Tanaka, H.; Osada, H.; Tomida, S.; Tatematsu, Y.; Yanagisawa, K.; Yatabe, Y.; Takamizawa, J.;Miyoshi, S.; Mitsudomi, T.; et al. Reduced expression of Dicer associated with poor prognosis in lung cancerpatients. Cancer Sci. 2005, 96, 111–115. [CrossRef] [PubMed]

Cancers 2020, 12, 1852 14 of 21

14. Lewis, B.P.; Burge, C.B.; Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates thatthousands of human genes are microRNA targets. Cell 2005, 120, 15–20. [CrossRef] [PubMed]

15. Friedman, R.C.; Farh, K.K.; Burge, C.B.; Bartel, D.P. Most mammalian mRNAs are conserved targets ofmicroRNAs. Genome Res. 2009, 19, 92–105. [CrossRef]

16. Ameres, S.L.; Zamore, P.D. Diversifying microRNA sequence and function. Nat. Rev. Mol. Cell Biol. 2013, 14,475–488. [CrossRef]

17. Hinske, L.C.; Franca, G.S.; Torres, H.A.; Ohara, D.T.; Lopes-Ramos, C.M.; Heyn, J.; Reis, L.F.;Ohno-Machado, L.; Kreth, S.; Galante, P.A. miRIAD-integrating microRNA inter- and intragenic data.Database (Oxf.) 2014, 2014. [CrossRef]

18. Misiewicz-Krzeminska, I.; Krzeminski, P.; Corchete, L.A.; Quwaider, D.; Rojas, E.A.; Herrero, A.B.;Gutierrez, N.C. Factors Regulating microRNA Expression and Function in Multiple Myeloma. Noncoding RNA2019, 5, 9. [CrossRef]

19. Gulyaeva, L.F.; Kushlinskiy, N.E. Regulatory mechanisms of microRNA expression. J. Transl. Med. 2016, 14, 143.[CrossRef]

20. Bail, S.; Swerdel, M.; Liu, H.; Jiao, X.; Goff, L.A.; Hart, R.P.; Kiledjian, M. Differential regulation of microRNAstability. RNA 2010, 16, 1032–1039. [CrossRef]

21. Harada, M.; Pokrovskaja-Tamm, K.; Soderhall, S.; Heyman, M.; Grander, D.; Corcoran, M. Involvement ofmiR17 pathway in glucocorticoid-induced cell death in pediatric acute lymphoblastic leukemia.Leuk Lymphoma 2012, 53, 2041–2050. [CrossRef] [PubMed]

22. Lin, S.; Gregory, R.I. MicroRNA biogenesis pathways in cancer. Nat. Rev. Cancer 2015, 15, 321–333. [CrossRef]23. Whiteside, T.L. The tumor microenvironment and its role in promoting tumor growth. Oncogene 2008, 27,

5904–5912. [CrossRef] [PubMed]24. Bhome, R.; Goh, R.W.; Bullock, M.D.; Pillar, N.; Thirdborough, S.M.; Mellone, M.; Mirnezami, R.; Galea, D.;

Veselkov, K.; Gu, Q.; et al. Exosomal microRNAs derived from colorectal cancer-associated fibroblasts:Role in driving cancer progression. Aging (Albany NY) 2017, 9, 2666–2694. [CrossRef]

25. Donnarumma, E.; Fiore, D.; Nappa, M.; Roscigno, G.; Adamo, A.; Iaboni, M.; Russo, V.; Affinito, A.; Puoti, I.;Quintavalle, C.; et al. Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressivephenotype in breast cancer. Oncotarget 2017, 8, 19592–19608. [CrossRef] [PubMed]

26. Richards, K.E.; Zeleniak, A.E.; Fishel, M.L.; Wu, J.; Littlepage, L.E.; Hill, R. Cancer-associated fibroblastexosomes regulate survival and proliferation of pancreatic cancer cells. Oncogene 2017, 36, 1770–1778.[CrossRef] [PubMed]

27. Au Yeung, C.L.; Co, N.N.; Tsuruga, T.; Yeung, T.L.; Kwan, S.Y.; Leung, C.S.; Li, Y.; Lu, E.S.; Kwan, K.;Wong, K.K.; et al. Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancercells through targeting APAF1. Nat. Commun. 2016, 7, 11150. [CrossRef]

28. Kohlhapp, F.J.; Mitra, A.K.; Lengyel, E.; Peter, M.E. MicroRNAs as mediators and communicators betweencancer cells and the tumor microenvironment. Oncogene 2015, 34, 5857–5868. [CrossRef]

29. Berindan-Neagoe, I.; Monroig, P.e.C.; Pasculli, B.; Calin, G.A. MicroRNAome genome: A treasure for cancerdiagnosis and therapy. CA Cancer J. Clin. 2014, 64, 311–336. [CrossRef]

30. Di Leva, G.; Garofalo, M.; Croce, C.M. MicroRNAs in cancer. Annu. Rev. Pathol. 2014, 9, 287–314. [CrossRef]31. Shah, M.Y.; Ferrajoli, A.; Sood, A.K.; Lopez-Berestein, G.; Calin, G.A. microRNA Therapeutics in Cancer-An

Emerging Concept. EBioMedicine 2016, 12, 34–42. [CrossRef] [PubMed]32. Lan, H.; Lu, H.; Wang, X.; Jin, H. MicroRNAs as potential biomarkers in cancer: Opportunities and challenges.

Biomed. Res. Int. 2015, 2015, 125094. [CrossRef] [PubMed]33. Lu, J.; Getz, G.; Miska, E.A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B.L.;

Mak, R.H.; Ferrando, A.A.; et al. MicroRNA expression profiles classify human cancers. Nature 2005, 435,834–838. [CrossRef]

34. Zhang, B.; Pan, X.; Cobb, G.P.; Anderson, T.A. microRNAs as oncogenes and tumor suppressors. Dev. Biol.2007, 302, 1–12. [CrossRef] [PubMed]

35. Iqbal, J.; Shen, Y.; Huang, X.; Liu, Y.; Wake, L.; Liu, C.; Deffenbacher, K.; Lachel, C.M.; Wang, C.; Rohr, J.;et al. Global microRNA expression profiling uncovers molecular markers for classification and prognosis inaggressive B-cell lymphoma. Blood 2015, 125, 1137–1145. [CrossRef]

Cancers 2020, 12, 1852 15 of 21

36. Caramuta, S.; Lee, L.; Ozata, D.M.; Akçakaya, P.; Georgii-Hemming, P.; Xie, H.; Amini, R.M.; Lawrie, C.H.;Enblad, G.; Larsson, C.; et al. Role of microRNAs and microRNA machinery in the pathogenesis of diffuselarge B-cell lymphoma. Blood Cancer J. 2013, 3, e152. [CrossRef]

37. Xiao, C.; Calado, D.P.; Galler, G.; Thai, T.H.; Patterson, H.C.; Wang, J.; Rajewsky, N.; Bender, T.P.; Rajewsky, K.MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 2007, 131, 146–159.[CrossRef]

38. Li, Y.; Cai, B.; Shen, L.; Dong, Y.; Lu, Q.; Sun, S.; Liu, S.; Ma, S.; Ma, P.X.; Chen, J. MiRNA-29b suppresses tumorgrowth through simultaneously inhibiting angiogenesis and tumorigenesis by targeting Akt3. Cancer Lett.2017, 397, 111–119. [CrossRef]

39. Croce, C.M. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 2009, 10,704–714. [CrossRef]

40. Esquela-Kerscher, A.; Slack, F.J. Oncomirs-microRNAs with a role in cancer. Nat. Rev. Cancer 2006, 6, 259–269.[CrossRef]

41. Hosseinahli, N.; Aghapour, M.; Duijf, P.H.G.; Baradaran, B. Treating cancer with microRNA replacementtherapy: A literature review. J. Cell Physiol. 2018, 233, 5574–5588. [CrossRef] [PubMed]

42. Osaki, M.; Takeshita, F.; Sugimoto, Y.; Kosaka, N.; Yamamoto, Y.; Yoshioka, Y.; Kobayashi, E.; Yamada, T.;Kawai, A.; Inoue, T.; et al. MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrixmetalloprotease-13 expression. Mol. Ther. 2011, 19, 1123–1130. [CrossRef]

43. Qiu, T.; Zhou, X.; Wang, J.; Du, Y.; Xu, J.; Huang, Z.; Zhu, W.; Shu, Y.; Liu, P. MiR-145, miR-133a and miR-133binhibit proliferation, migration, invasion and cell cycle progression via targeting transcription factor Sp1 ingastric cancer. FEBS Lett. 2014, 588, 1168–1177. [CrossRef] [PubMed]

44. Bader, A.G.; Brown, D.; Winkler, M. The promise of microRNA replacement therapy. Cancer Res. 2010, 70,7027–7030. [CrossRef] [PubMed]

45. Broderick, J.A.; Zamore, P.D. MicroRNA therapeutics. Gene Ther. 2011, 18, 1104–1110. [CrossRef] [PubMed]46. Kong, Y.W.; Ferland-McCollough, D.; Jackson, T.J.; Bushell, M. microRNAs in cancer management.

Lancet Oncol. 2012, 13, e249–e258. [CrossRef]47. Lu, Z.; He, Q.; Liang, J.; Li, W.; Su, Q.; Chen, Z.; Wan, Q.; Zhou, X.; Cao, L.; Sun, J.; et al. miR-31-5p Is a

Potential Circulating Biomarker and Therapeutic Target for Oral Cancer. Mol. Ther. Nucleic Acids 2019, 16,471–480. [CrossRef]

48. Kao, Y.Y.; Chou, C.H.; Yeh, L.Y.; Chen, Y.F.; Chang, K.W.; Liu, C.J.; Fan Chiang, C.Y.; Lin, S.C. MicroRNAmiR-31 targets SIRT3 to disrupt mitochondrial activity and increase oxidative stress in oral carcinoma.Cancer Lett. 2019, 456, 40–48. [CrossRef]

49. Guo, H.; Ji, F.; Zhao, X.; Yang, X.; He, J.; Huang, L.; Zhang, Y. MicroRNA-371a-3p promotes progression ofgastric cancer by targeting TOB1. Cancer Lett. 2019, 443, 179–188. [CrossRef]

50. Dieckmann, K.P.; Spiekermann, M.; Balks, T.; Flor, I.; Löning, T.; Bullerdiek, J.; Belge, G. MicroRNAsmiR-371-3 in serum as diagnostic tools in the management of testicular germ cell tumours. Br. J. Cancer2012, 107, 1754–1760. [CrossRef]

51. Yanaihara, N.; Caplen, N.; Bowman, E.; Seike, M.; Kumamoto, K.; Yi, M.; Stephens, R.M.; Okamoto, A.;Yokota, J.; Tanaka, T.; et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell 2006, 9, 189–198. [CrossRef] [PubMed]

52. Kong, Y.; Zou, S.; Yang, F.; Xu, X.; Bu, W.; Jia, J.; Liu, Z. RUNX3-mediated up-regulation of miR-29b suppressesthe proliferation and migration of gastric cancer cells by targeting KDM2A. Cancer Lett. 2016, 381, 138–148.[CrossRef] [PubMed]

53. Yang, S.; Li, Y.; Gao, J.; Zhang, T.; Li, S.; Luo, A.; Chen, H.; Ding, F.; Wang, X.; Liu, Z. MicroRNA-34 suppressesbreast cancer invasion and metastasis by directly targeting Fra-1. Oncogene 2013, 32, 4294–4303. [CrossRef]

54. Ma, W.; Xiao, G.G.; Mao, J.; Lu, Y.; Song, B.; Wang, L.; Fan, S.; Fan, P.; Hou, Z.; Li, J.; et al. Dysregulation ofthe miR-34a-SIRT1 axis inhibits breast cancer stemness. Oncotarget 2015, 6, 10432–10444. [CrossRef]

55. Beg, M.S.; Brenner, A.J.; Sachdev, J.; Borad, M.; Kang, Y.K.; Stoudemire, J.; Smith, S.; Bader, A.G.; Kim, S.;Hong, D.S. Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients withadvanced solid tumors. Invest. New Drugs 2017, 35, 180–188. [CrossRef]

56. Zhang, X.J.; Ye, H.; Zeng, C.W.; He, B.; Zhang, H.; Chen, Y.Q. Dysregulation of miR-15a and miR-214 inhuman pancreatic cancer. J. Hematol. Oncol. 2010, 3, 46. [CrossRef] [PubMed]

Cancers 2020, 12, 1852 16 of 21

57. Bonci, D.; Coppola, V.; Musumeci, M.; Addario, A.; Giuffrida, R.; Memeo, L.; D’Urso, L.; Pagliuca, A.;Biffoni, M.; Labbaye, C.; et al. The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multipleoncogenic activities. Nat. Med. 2008, 14, 1271–1277. [CrossRef] [PubMed]

58. Elmén, J.; Lindow, M.; Schütz, S.; Lawrence, M.; Petri, A.; Obad, S.; Lindholm, M.; Hedtjärn, M.; Hansen, H.F.;Berger, U.; et al. LNA-mediated microRNA silencing in non-human primates. Nature 2008, 452, 896–899.[CrossRef] [PubMed]

59. Garzon, R.; Marcucci, G.; Croce, C.M. Targeting microRNAs in cancer: Rationale, strategies and challenges.Nat. Rev. Drug Discov. 2010, 9, 775–789. [CrossRef]

60. Monroig, P.e.C.; Chen, L.; Zhang, S.; Calin, G.A. Small molecule compounds targeting miRNAs for cancertherapy. Adv. Drug Deliv. Rev. 2015, 81, 104–116. [CrossRef]

61. Vester, B.; Wengel, J. LNA (locked nucleic acid): High-affinity targeting of complementary RNA and DNA.Biochemistry 2004, 43, 13233–13241. [CrossRef] [PubMed]

62. Krützfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K.G.; Tuschl, T.; Manoharan, M.; Stoffel, M. Silencing ofmicroRNAs in vivo with ‘antagomirs’. Nature 2005, 438, 685–689. [CrossRef] [PubMed]

63. Han, M.; Liu, M.; Wang, Y.; Chen, X.; Xu, J.; Sun, Y.; Zhao, L.; Qu, H.; Fan, Y.; Wu, C. Antagonism ofmiR-21 reverses epithelial-mesenchymal transition and cancer stem cell phenotype through AKT/ERK1/2inactivation by targeting PTEN. PLoS ONE 2012, 7, e39520. [CrossRef] [PubMed]

64. Liu, L.Z.; Li, C.; Chen, Q.; Jing, Y.; Carpenter, R.; Jiang, Y.; Kung, H.F.; Lai, L.; Jiang, B.H. MiR-21 inducedangiogenesis through AKT and ERK activation and HIF-1α expression. PLoS ONE 2011, 6, e19139. [CrossRef][PubMed]

65. Chen, Y.; Gao, D.Y.; Huang, L. In vivo delivery of miRNAs for cancer therapy: Challenges and strategies.Adv. Drug Deliv. Rev. 2015, 81, 128–141. [CrossRef]

66. Chen, L.; Zhang, K.; Shi, Z.; Zhang, A.; Jia, Z.; Wang, G.; Pu, P.; Kang, C.; Han, L. A lentivirus-mediatedmiR-23b sponge diminishes the malignant phenotype of glioma cells in vitro and in vivo. Oncol. Rep.2014, 31, 1573–1580. [CrossRef]

67. Jansson, M.D.; Lund, A.H. MicroRNA and cancer. Mol. Oncol. 2012, 6, 590–610. [CrossRef]68. Kota, J.; Chivukula, R.R.; O’Donnell, K.A.; Wentzel, E.A.; Montgomery, C.L.; Hwang, H.W.; Chang, T.C.;

Vivekanandan, P.; Torbenson, M.; Clark, K.R.; et al. Therapeutic microRNA delivery suppresses tumorigenesisin a murine liver cancer model. Cell 2009, 137, 1005–1017. [CrossRef] [PubMed]

69. Stylianopoulos, T.; Jain, R.K. Combining two strategies to improve perfusion and drug delivery in solidtumors. Proc. Natl. Acad. Sci. USA 2013, 110, 18632–18637. [CrossRef] [PubMed]

70. Kleger, A.; Perkhofer, L.; Seufferlein, T. Smarter drugs emerging in pancreatic cancer therapy. Ann. Oncol.2014, 25, 1260–1270. [CrossRef] [PubMed]

71. Castelli, D.D.; Terreno, E.; Cabella, C.; Chaabane, L.; Lanzardo, S.; Tei, L.; Visigalli, M.; Aime, S. Evidence forin vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes. NMR Biomed. 2009, 22,1084–1092. [CrossRef] [PubMed]

72. Wu, Y.; Crawford, M.; Mao, Y.; Lee, R.J.; Davis, I.C.; Elton, T.S.; Lee, L.J.; Nana-Sinkam, S.P. TherapeuticDelivery of MicroRNA-29b by Cationic Lipoplexes for Lung Cancer. Mol. Ther. Nucleic Acids 2013, 2, e84.[CrossRef] [PubMed]

73. Rai, K.; Takigawa, N.; Ito, S.; Kashihara, H.; Ichihara, E.; Yasuda, T.; Shimizu, K.; Tanimoto, M.; Kiura, K.Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosinekinase inhibitor-resistance in lung cancer cells. Mol. Cancer Ther. 2011, 10, 1720–1727. [CrossRef]

74. Wiggins, J.F.; Ruffino, L.; Kelnar, K.; Omotola, M.; Patrawala, L.; Brown, D.; Bader, A.G. Development ofa lung cancer therapeutic based on the tumor suppressor microRNA-34. Cancer Res. 2010, 70, 5923–5930.[CrossRef] [PubMed]

75. Trang, P.; Wiggins, J.F.; Daige, C.L.; Cho, C.; Omotola, M.; Brown, D.; Weidhaas, J.B.; Bader, A.G.; Slack, F.J.Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lungtumors in mice. Mol. Ther. 2011, 19, 1116–1122. [CrossRef] [PubMed]

76. Cortez, M.A.; Valdecanas, D.; Zhang, X.; Zhan, Y.; Bhardwaj, V.; Calin, G.A.; Komaki, R.; Giri, D.K.; Quini, C.C.;Wolfe, T.; et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer. Mol. Ther. 2014, 22,1494–1503. [CrossRef]

Cancers 2020, 12, 1852 17 of 21

77. Hu, Q.; Wang, K.; Sun, X.; Li, Y.; Fu, Q.; Liang, T.; Tang, G. A redox-sensitive, oligopeptide-guided,self-assembling, and efficiency-enhanced (ROSE) system for functional delivery of microRNA therapeuticsfor treatment of hepatocellular carcinoma. Biomaterials 2016, 104, 192–200. [CrossRef]

78. Yang, Y.P.; Chien, Y.; Chiou, G.Y.; Cherng, J.Y.; Wang, M.L.; Lo, W.L.; Chang, Y.L.; Huang, P.I.; Chen, Y.W.;Shih, Y.H.; et al. Inhibition of cancer stem cell-like properties and reduced chemoradioresistance ofglioblastoma using microRNA145 with cationic polyurethane-short branch PEI. Biomaterials 2012, 33,1462–1476. [CrossRef]

79. Ibrahim, A.F.; Weirauch, U.; Thomas, M.; Grünweller, A.; Hartmann, R.K.; Aigner, A. MicroRNA replacementtherapy for miR-145 and miR-33a is efficacious in a model of colon carcinoma. Cancer Res. 2011, 71, 5214–5224.[CrossRef]

80. Mittal, A.; Chitkara, D.; Behrman, S.W.; Mahato, R.I. Efficacy of gemcitabine conjugated and miRNA-205complexed micelles for treatment of advanced pancreatic cancer. Biomaterials 2014, 35, 7077–7087. [CrossRef]

81. Qian, X.; Long, L.; Shi, Z.; Liu, C.; Qiu, M.; Sheng, J.; Pu, P.; Yuan, X.; Ren, Y.; Kang, C. Star-branchedamphiphilic PLA-b-PDMAEMA copolymers for co-delivery of miR-21 inhibitor and doxorubicin to treatglioma. Biomaterials 2014, 35, 2322–2335. [CrossRef] [PubMed]

82. Seo, Y.E.; Suh, H.W.; Bahal, R.; Josowitz, A.; Zhang, J.; Song, E.; Cui, J.; Noorbakhsh, S.; Jackson, C.; Bu, T.;et al. Nanoparticle-mediated intratumoral inhibition of miR-21 for improved survival in glioblastoma.Biomaterials 2019, 201, 87–98. [CrossRef] [PubMed]

83. Sonkoly, E.; Lovén, J.; Xu, N.; Meisgen, F.; Wei, T.; Brodin, P.; Jaks, V.; Kasper, M.; Shimokawa, T.; Harada, M.;et al. MicroRNA-203 functions as a tumor suppressor in basal cell carcinoma. Oncogenesis 2012, 1, e3.[CrossRef] [PubMed]

84. Morimoto, Y.; Mizushima, T.; Wu, X.; Okuzaki, D.; Yokoyama, Y.; Inoue, A.; Hata, T.; Hirose, H.; Qian, Y.;Wang, J.; et al. miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 andTFDP1 in colon cancer cells. Br. J. Cancer 2020, 122, 1037–1049. [CrossRef]

85. Hiraki, M.; Nishimura, J.; Takahashi, H.; Wu, X.; Takahashi, Y.; Miyo, M.; Nishida, N.; Uemura, M.; Hata, T.;Takemasa, I.; et al. Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment AgainstMutant KRAS Colorectal Cancer. Mol. Ther. Nucleic Acids 2015, 4, e231. [CrossRef]

86. Inoue, A.; Mizushima, T.; Wu, X.; Okuzaki, D.; Kambara, N.; Ishikawa, S.; Wang, J.; Qian, Y.; Hirose, H.;Yokoyama, Y.; et al. A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities viaInhibition of NF-κB Signaling in. Mol. Cancer Ther. 2018, 17, 977–987. [CrossRef]

87. Akao, Y.; Iio, A.; Itoh, T.; Noguchi, S.; Itoh, Y.; Ohtsuki, Y.; Naoe, T. Microvesicle-mediated RNA moleculedelivery system using monocytes/macrophages. Mol. Ther. 2011, 19, 395–399. [CrossRef]

88. Katakowski, M.; Buller, B.; Zheng, X.; Lu, Y.; Rogers, T.; Osobamiro, O.; Shu, W.; Jiang, F.; Chopp, M.Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett. 2013, 335,201–204. [CrossRef]

89. Vázquez-Ríos, A.J.; Molina-Crespo, Á.; Bouzo, B.L.; López-López, R.; Moreno-Bueno, G.; de la Fuente, M.Exosome-mimetic nanoplatforms for targeted cancer drug delivery. J. Nanobiotechnol. 2019, 17, 85. [CrossRef]

90. Lou, G.; Song, X.; Yang, F.; Wu, S.; Wang, J.; Chen, Z.; Liu, Y. Exosomes derived from miR-122-modifiedadipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma. J. Hematol. Oncol.2015, 8, 122. [CrossRef] [PubMed]

91. Ohno, S.; Takanashi, M.; Sudo, K.; Ueda, S.; Ishikawa, A.; Matsuyama, N.; Fujita, K.; Mizutani, T.; Ohgi, T.;Ochiya, T.; et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breastcancer cells. Mol. Ther. 2013, 21, 185–191. [CrossRef] [PubMed]

92. Tazawa, H.; Tsuchiya, N.; Izumiya, M.; Nakagama, H. Tumor-suppressive miR-34a induces senescence-likegrowth arrest through modulation of the E2F pathway in human colon cancer cells. Proc. Natl. Acad. Sci. USA2007, 104, 15472–15477. [CrossRef] [PubMed]

93. Takeshita, F.; Patrawala, L.; Osaki, M.; Takahashi, R.U.; Yamamoto, Y.; Kosaka, N.; Kawamata, M.; Kelnar, K.;Bader, A.G.; Brown, D.; et al. Systemic delivery of synthetic microRNA-16 inhibits the growth of metastaticprostate tumors via downregulation of multiple cell-cycle genes. Mol. Ther. 2010, 18, 181–187. [CrossRef][PubMed]

94. Hao, Z.; Fan, W.; Hao, J.; Wu, X.; Zeng, G.Q.; Zhang, L.J.; Nie, S.F.; Wang, X.D. Efficient delivery of microRNA to bone-metastatic prostate tumors by using aptamer-conjugated atelocollagen in vitro and in vivo.Drug Deliv. 2016, 23, 874–881. [CrossRef]

Cancers 2020, 12, 1852 18 of 21

95. Halle, B.; Marcusson, E.G.; Aaberg-Jessen, C.; Jensen, S.S.; Meyer, M.; Schulz, M.K.; Andersen, C.;Kristensen, B.W. Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stabilityand causes efficient target de-repression: A proof of concept. J. Neurooncol. 2016, 126, 47–55. [CrossRef]

96. Trang, P.; Medina, P.P.; Wiggins, J.F.; Ruffino, L.; Kelnar, K.; Omotola, M.; Homer, R.; Brown, D.; Bader, A.G.;Weidhaas, J.B.; et al. Regression of murine lung tumors by the let-7 microRNA. Oncogene 2010, 29, 1580–1587.[CrossRef]

97. He, X.X.; Chang, Y.; Meng, F.Y.; Wang, M.Y.; Xie, Q.H.; Tang, F.; Li, P.Y.; Song, Y.H.; Lin, J.S. MicroRNA-375targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo.Oncogene 2012, 31, 3357–3369. [CrossRef]

98. Sureban, S.M.; May, R.; Mondalek, F.G.; Qu, D.; Ponnurangam, S.; Pantazis, P.; Anant, S.; Ramanujam, R.P.;Houchen, C.W. Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectalcancer tumor growth via a Notch-1 dependent mechanism. J. Nanobiotechnol. 2011, 9, 40. [CrossRef]

99. Aagaard, L.; Rossi, J.J. RNAi therapeutics: Principles, prospects and challenges. Adv. Drug Deliv. Rev.2007, 59, 75–86. [CrossRef]

100. Wang, X.; Yu, B.; Ren, W.; Mo, X.; Zhou, C.; He, H.; Jia, H.; Wang, L.; Jacob, S.T.; Lee, R.J.; et al. Enhanced hepaticdelivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations. J. Control. Release2013, 172, 690–698. [CrossRef]

101. Davis, S.; Lollo, B.; Freier, S.; Esau, C. Improved targeting of miRNA with antisense oligonucleotides.Nucleic Acids Res. 2006, 34, 2294–2304. [CrossRef] [PubMed]

102. Elmén, J.; Lindow, M.; Silahtaroglu, A.; Bak, M.; Christensen, M.; Lind-Thomsen, A.; Hedtjärn, M.; Hansen, J.B.;Hansen, H.F.; Straarup, E.M.; et al. Antagonism of microRNA-122 in mice by systemically administeredLNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res.2008, 36, 1153–1162. [CrossRef] [PubMed]

103. Stenvang, J.; Petri, A.; Lindow, M.; Obad, S.; Kauppinen, S. Inhibition of microRNA function by antimiRoligonucleotides. Silence 2012, 3, 1. [CrossRef] [PubMed]

104. Teplyuk, N.M.; Uhlmann, E.J.; Gabriely, G.; Volfovsky, N.; Wang, Y.; Teng, J.; Karmali, P.; Marcusson, E.;Peter, M.; Mohan, A.; et al. Therapeutic potential of targeting microRNA-10b in established intracranialglioblastoma: First steps toward the clinic. EMBO Mol. Med. 2016, 8, 268–287. [CrossRef] [PubMed]

105. Yang, N. An overview of viral and nonviral delivery systems for microRNA. Int. J. Pharm. Investig. 2015, 5,179–181. [CrossRef]

106. O’Neill, C.P.; Dwyer, R.M. Nanoparticle-Based Delivery of Tumor Suppressor microRNA for Cancer Therapy.Cells 2020, 9, 521. [CrossRef]

107. Herrera-Carrillo, E.; Liu, Y.P.; Berkhout, B. Improving miRNA Delivery by Optimizing miRNA ExpressionCassettes in Diverse Virus Vectors. Hum. Gene Ther. Methods 2017, 28, 177–190. [CrossRef] [PubMed]

108. Kasar, S.; Salerno, E.; Yuan, Y.; Underbayev, C.; Vollenweider, D.; Laurindo, M.F.; Fernandes, H.; Bonci, D.;Addario, A.; Mazzella, F.; et al. Systemic in vivo lentiviral delivery of miR-15a/16 reduces malignancy in theNZB de novo mouse model of chronic lymphocytic leukemia. Genes Immun. 2012, 13, 109–119. [CrossRef][PubMed]

109. Liu, Y.; Lai, L.; Chen, Q.; Song, Y.; Xu, S.; Ma, F.; Wang, X.; Wang, J.; Yu, H.; Cao, X.; et al. MicroRNA-494is required for the accumulation and functions of tumor-expanded myeloid-derived suppressor cells viatargeting of PTEN. J. Immunol. 2012, 188, 5500–5510. [CrossRef]

110. Yin, H.; Kanasty, R.L.; Eltoukhy, A.A.; Vegas, A.J.; Dorkin, J.R.; Anderson, D.G. Non-viral vectors forgene-based therapy. Nat. Rev. Genet. 2014, 15, 541–555. [CrossRef]

111. Bai, Z.; Wei, J.; Yu, C.; Han, X.; Qin, X.; Zhang, C.; Liao, W.; Li, L.; Huang, W. Non-viral nanocarriers forintracellular delivery of microRNA therapeutics. J. Mater. Chem. B 2019, 7, 1209–1225. [CrossRef] [PubMed]

112. Wang, H.; Liu, S.; Jia, L.; Chu, F.; Zhou, Y.; He, Z.; Guo, M.; Chen, C.; Xu, L. Nanostructured lipid carriers forMicroRNA delivery in tumor gene therapy. Cancer Cell Int. 2018, 18, 101. [CrossRef] [PubMed]

113. Bakhshandeh, B.; Soleimani, M.; Hafizi, M.; Ghaemi, N. A comparative study on nonviral genetic modificationsin cord blood and bone marrow mesenchymal stem cells. Cytotechnology 2012, 64, 523–540. [CrossRef]

114. Vaughan, H.J.; Green, J.J.; Tzeng, S.Y. Cancer-Targeting Nanoparticles for Combinatorial Nucleic AcidDelivery. Adv. Mater. 2020, 32, e1901081. [CrossRef]

115. Mintzer, M.A.; Simanek, E.E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109, 259–302. [CrossRef][PubMed]

Cancers 2020, 12, 1852 19 of 21

116. Wang, A.Z.; Langer, R.; Farokhzad, O.C. Nanoparticle delivery of cancer drugs. Annu. Rev. Med. 2012, 63,185–198. [CrossRef]

117. Pecot, C.V.; Calin, G.A.; Coleman, R.L.; Lopez-Berestein, G.; Sood, A.K. RNA interference in the clinic:Challenges and future directions. Nat. Rev. Cancer 2011, 11, 59–67. [CrossRef]

118. Blanco, E.; Shen, H.; Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drugdelivery. Nat. Biotechnol. 2015, 33, 941–951. [CrossRef]

119. Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M.;Miyazono, K.; Uesaka, M.; et al. Accumulation of sub-100 nm polymeric micelles in poorly permeabletumours depends on size. Nat. Nanotechnol. 2011, 6, 815–823. [CrossRef]

120. Torchilin, V.P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 2005, 4,145–160. [CrossRef]

121. Ait-Oudhia, S.; Mager, D.E.; Straubinger, R.M. Application of pharmacokinetic and pharmacodynamicanalysis to the development of liposomal formulations for oncology. Pharmaceutics 2014, 6, 137–174.[CrossRef]

122. Sun, X.; Yan, X.; Jacobson, O.; Sun, W.; Wang, Z.; Tong, X.; Xia, Y.; Ling, D.; Chen, X. Improved Tumor Uptakeby Optimizing Liposome Based RES Blockade Strategy. Theranostics 2017, 7, 319–328. [CrossRef] [PubMed]

123. Hong, D.S.; Kang, Y.K.; Borad, M.; Sachdev, J.; Ejadi, S.; Lim, H.Y.; Brenner, A.J.; Park, K.; Lee, J.L.; Kim, T.Y.;et al. Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.Br. J. Cancer 2020, -1. [CrossRef]

124. Fernandez, C.A.; Rice, K.G. Engineered nanoscaled polyplex gene delivery systems. Mol. Pharm. 2009, 6,1277–1289. [CrossRef]

125. Duncan, R.; Izzo, L. Dendrimer biocompatibility and toxicity. Adv. Drug Deliv. Rev. 2005, 57, 2215–2237.[CrossRef]

126. Mao, S.; Sun, W.; Kissel, T. Chitosan-based formulations for delivery of DNA and siRNA. Adv. Drug Deliv. Rev.2010, 62, 12–27. [CrossRef] [PubMed]

127. Kaban, K.; Salva, E.; Akbuga, J. The effects of chitosan/miR-200c nanoplexes on different stages of cancers inbreast cancer cell lines. Eur. J. Pharm. Sci. 2016, 95, 103–110. [CrossRef] [PubMed]

128. Kaban, K.; Salva, E.; Akbuga, J. In Vitro Dose Studies on Chitosan Nanoplexes for microRNA Delivery inBreast Cancer Cells. Nucleic Acid Ther. 2017, 27, 45–55. [CrossRef]

129. Gaur, S.; Wen, Y.; Song, J.H.; Parikh, N.U.; Mangala, L.S.; Blessing, A.M.; Ivan, C.; Wu, S.Y.; Varkaris, A.;Shi, Y.; et al. Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in thebone and its expression induces non-canonical autophagy. Oncotarget 2015, 6, 29161–29177. [CrossRef]

130. Dailey, L.A.; Wittmar, M.; Kissel, T. The role of branched polyesters and their modifications in the developmentof modern drug delivery vehicles. J. Control. Release 2005, 101, 137–149. [CrossRef] [PubMed]

131. Tinsley-Bown, A.M.; Fretwell, R.; Dowsett, A.B.; Davis, S.L.; Farrar, G.H. Formulation ofpoly(D,L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. J. Control. Release 2000, 66,229–241. [CrossRef]

132. Liang, G.; Zhu, Y.; Jing, A.; Wang, J.; Hu, F.; Feng, W.; Xiao, Z.; Chen, B. Cationic microRNA-deliveringnanocarriers for efficient treatment of colon carcinoma in xenograft model. Gene Ther. 2016, 23, 829–838.[CrossRef]

133. Cosco, D.; Cilurzo, F.; Maiuolo, J.; Federico, C.; Di Martino, M.T.; Cristiano, M.C.; Tassone, P.; Fresta, M.;Paolino, D. Delivery of miR-34a by chitosan/PLGA nanoplexes for the anticancer treatment of multiplemyeloma. Sci. Rep. 2015, 5, 17579. [CrossRef]

134. Zhou, K.; Nguyen, L.H.; Miller, J.B.; Yan, Y.; Kos, P.; Xiong, H.; Li, L.; Hao, J.; Minnig, J.T.; Zhu, H.; et al.Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model.Proc. Natl. Acad. Sci. USA 2016, 113, 520–525. [CrossRef]

135. Shatsberg, Z.; Zhang, X.; Ofek, P.; Malhotra, S.; Krivitsky, A.; Scomparin, A.; Tiram, G.; Calderón, M.;Haag, R.; Satchi-Fainaro, R. Functionalized nanogels carrying an anticancer microRNA for glioblastomatherapy. J. Control. Release 2016, 239, 159–168. [CrossRef] [PubMed]

136. Sokolova, V.; Epple, M. Inorganic nanoparticles as carriers of nucleic acids into cells. Angew. Chem. Int.Ed. Engl. 2008, 47, 1382–1395. [CrossRef] [PubMed]

Cancers 2020, 12, 1852 20 of 21

137. Gilam, A.; Conde, J.; Weissglas-Volkov, D.; Oliva, N.; Friedman, E.; Artzi, N.; Shomron, N. Local microRNAdelivery targets Palladin and prevents metastatic breast cancer. Nat. Commun. 2016, 7, 12868. [CrossRef][PubMed]

138. Lee, T.J.; Yoo, J.Y.; Shu, D.; Li, H.; Zhang, J.; Yu, J.G.; Jaime-Ramirez, A.C.; Acunzo, M.; Romano, G.; Cui, R.;et al. RNA Nanoparticle-Based Targeted Therapy for Glioblastoma through Inhibition of Oncogenic miR-21.Mol. Ther. 2017, 25, 1544–1555. [CrossRef]

139. Bertucci, A.; Prasetyanto, E.A.; Septiadi, D.; Manicardi, A.; Brognara, E.; Gambari, R.; Corradini, R.; De Cola, L.Combined Delivery of Temozolomide and Anti-miR221 PNA Using Mesoporous Silica Nanoparticles InducesApoptosis in Resistant Glioma Cells. Small 2015, 11, 5687–5695. [CrossRef]

140. Ochiya, T.; Nagahara, S.; Sano, A.; Itoh, H.; Terada, M. Biomaterials for gene delivery: Atelocollagen-mediatedcontrolled release of molecular medicines. Curr. Gene Ther. 2001, 1, 31–52. [CrossRef]

141. Tkach, M.; Théry, C. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go.Cell 2016, 164, 1226–1232. [CrossRef] [PubMed]

142. Simpson, R.J.; Jensen, S.S.; Lim, J.W. Proteomic profiling of exosomes: Current perspectives. Proteomics2008, 8, 4083–4099. [CrossRef] [PubMed]

143. EL Andaloussi, S.; Mäger, I.; Breakefield, X.O.; Wood, M.J. Extracellular vesicles: Biology and emergingtherapeutic opportunities. Nat. Rev. Drug Discov. 2013, 12, 347–357. [CrossRef]

144. Fuhrmann, G.; Herrmann, I.; Stevens, M. Cell-derived vesicles for drug therapy and diagnostics:Opportunities and challenges. Nano Today 2015, 10, 397–409. [CrossRef] [PubMed]

145. Rupaimoole, R.; Slack, F.J. MicroRNA therapeutics: Towards a new era for the management of cancer andother diseases. Nat. Rev. Drug Discov. 2017, 16, 203–222. [CrossRef] [PubMed]

146. Kalluri, R.; LeBleu, V.S. The biology, function, and biomedical applications of exosomes. Science 2020, 367.[CrossRef] [PubMed]

147. Hessvik, N.P.; Llorente, A. Current knowledge on exosome biogenesis and release. Cell Mol. Life Sci.2018, 75, 193–208. [CrossRef]

148. Stahl, P.D.; Raposo, G. Exosomes and extracellular vesicles: The path forward. Essays Biochem. 2018, 62,119–124. [CrossRef]

149. Thery, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.;Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018(MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of theMISEV2014 guidelines. J. Extracell Vesicles 2018, 7, 1535750. [CrossRef]

150. Valadi, H.; Ekström, K.; Bossios, A.; Sjöstrand, M.; Lee, J.J.; Lötvall, J.O. Exosome-mediated transfer ofmRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9,654–659. [CrossRef]

151. Skog, J.; Würdinger, T.; van Rijn, S.; Meijer, D.H.; Gainche, L.; Sena-Esteves, M.; Curry, W.T.; Carter, B.S.;Krichevsky, A.M.; Breakefield, X.O. Glioblastoma microvesicles transport RNA and proteins that promotetumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 2008, 10, 1470–1476. [CrossRef] [PubMed]

152. Chalmin, F.; Ladoire, S.; Mignot, G.; Vincent, J.; Bruchard, M.; Remy-Martin, J.; Boireau, W.; Rouleau, A.;Simon, B.; Lanneau, D.; et al. Membrane-associated Hsp72 from tumor-derived exosomes mediatesSTAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells.J. Clin. Investig. 2010, 120, 457–471. [CrossRef] [PubMed]

153. Mathieu, M.; Martin-Jaular, L.; Lavieu, G.; Théry, C. Specificities of secretion and uptake of exosomes andother extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 2019, 21, 9–17. [CrossRef] [PubMed]

154. Vickers, K.C.; Palmisano, B.T.; Shoucri, B.M.; Shamburek, R.D.; Remaley, A.T. MicroRNAs are transportedin plasma and delivered to recipient cells by high-density lipoproteins. Nat. Cell Biol. 2011, 13, 423–433.[CrossRef]

155. Zhuang, X.; Xiang, X.; Grizzle, W.; Sun, D.; Zhang, S.; Axtell, R.C.; Ju, S.; Mu, J.; Zhang, L.; Steinman, L.; et al.Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugsfrom the nasal region to the brain. Mol. Ther. 2011, 19, 1769–1779. [CrossRef]

156. Liu, C.; Su, C. Design strategies and application progress of therapeutic exosomes. Theranostics 2019, 9,1015–1028. [CrossRef] [PubMed]

Cancers 2020, 12, 1852 21 of 21

157. Kooijmans, S.A.A.; Fliervoet, L.A.L.; van der Meel, R.; Fens, M.H.A.M.; Heijnen, H.F.G.; van Bergen EnHenegouwen, P.M.P.; Vader, P.; Schiffelers, R.M. PEGylated and targeted extracellular vesicles displayenhanced cell specificity and circulation time. J. Control. Release 2016, 224, 77–85. [CrossRef]

158. Guessous, F.; Alvarado-Velez, M.; Marcinkiewicz, L.; Zhang, Y.; Kim, J.; Heister, S.; Kefas, B.; Godlewski, J.;Schiff, D.; Purow, B.; et al. Oncogenic effects of miR-10b in glioblastoma stem cells. J. Neurooncol. 2013, 112,153–163. [CrossRef]

159. Nicoloso, M.S.; Spizzo, R.; Shimizu, M.; Rossi, S.; Calin, G.A. MicroRNAs–the micro steering wheel of tumourmetastases. Nat. Rev. Cancer 2009, 9, 293–302. [CrossRef]

160. Ma, L.; Reinhardt, F.; Pan, E.; Soutschek, J.; Bhat, B.; Marcusson, E.G.; Teruya-Feldstein, J.; Bell, G.W.;Weinberg, R.A. Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model.Nat. Biotechnol. 2010, 28, 341–347. [CrossRef]

161. Sheedy, P.; Medarova, Z. The fundamental role of miR-10b in metastatic cancer. Am. J. Cancer Res. 2018, 8,1674–1688. [PubMed]

162. Callegari, E.; Elamin, B.K.; Giannone, F.; Milazzo, M.; Altavilla, G.; Fornari, F.; Giacomelli, L.; D’Abundo, L.;Ferracin, M.; Bassi, C.; et al. Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model.Hepatology 2012, 56, 1025–1033. [CrossRef] [PubMed]

163. Gallo Cantafio, M.E.; Nielsen, B.S.; Mignogna, C.; Arbitrio, M.; Botta, C.; Frandsen, N.M.; Rolfo, C.;Tagliaferri, P.; Tassone, P.; Di Martino, M.T. Pharmacokinetics and Pharmacodynamics of a 13-merLNA-inhibitor-miR-221 in Mice and Non-human Primates. Mol. Ther. Nucleic Acids 2016, 5. [CrossRef][PubMed]

164. Bandi, N.; Zbinden, S.; Gugger, M.; Arnold, M.; Kocher, V.; Hasan, L.; Kappeler, A.; Brunner, T.; Vassella, E.miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequentlydeleted or down-regulated in non-small cell lung cancer. Cancer Res. 2009, 69, 5553–5559. [CrossRef][PubMed]

165. Reid, G.; Pel, M.E.; Kirschner, M.B.; Cheng, Y.Y.; Mugridge, N.; Weiss, J.; Williams, M.; Wright, C.; Edelman, J.J.;Vallely, M.P.; et al. Restoring expression of miR-16: A novel approach to therapy for malignant pleuralmesothelioma. Ann. Oncol. 2013, 24, 3128–3135. [CrossRef]

166. Ozcan, G.; Ozpolat, B.; Coleman, R.L.; Sood, A.K.; Lopez-Berestein, G. Preclinical and clinical developmentof siRNA-based therapeutics. Adv. Drug Deliv. Rev. 2015, 87, 108–119. [CrossRef]

167. Cortez, M.A.; Ivan, C.; Valdecanas, D.; Wang, X.; Peltier, H.J.; Ye, Y.; Araujo, L.; Carbone, D.P.; Shilo, K.;Giri, D.K.; et al. PDL1 Regulation by p53 via miR-34. J. Natl. Cancer Inst. 2016, 108. [CrossRef]

168. Reid, G.; Kao, S.C.; Pavlakis, N.; Brahmbhatt, H.; MacDiarmid, J.; Clarke, S.; Boyer, M.; van Zandwijk, N.Clinical development of TargomiRs, a miRNA mimic-based treatment for patients with recurrent thoraciccancer. Epigenomics 2016, 8, 1079–1085. [CrossRef]

169. Winata, P.; Williams, M.; McGowan, E.; Nassif, N.; van Zandwijk, N.; Reid, G. The analysis of novel microRNAmimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.BMC Res. Notes 2017, 10, 600. [CrossRef]

170. Rupaimoole, R.; Calin, G.A.; Lopez-Berestein, G.; Sood, A.K. miRNA Deregulation in Cancer Cells and theTumor Microenvironment. Cancer Discov. 2016, 6, 235–246. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).